Using Stable Isotope Techniques to Analyze the Trophic Relationship between Argentine Hake (Merluccius hubbsi) and Anisakidae

Simple Summary The Argentine hake (Merluccius hubbsi) is a significant fish species in the Southwest Atlantic, both economically and ecologically. These fish often host Anisakidae larvae, which are common parasites. Despite their prevalence, the specific nutritional relationships between these parasites and their host are not well understood. This study uses stable isotope techniques to investigate these relationships by analyzing the carbon and nitrogen compositions in different tissues of the hake and comparing them to those in the parasites. The results show significant differences, indicating that the parasites primarily obtain their nutrients from the hake’s stomach but also from other tissues. This study provides new insights into the complex nutritional interactions between the M. hubbsi and Anisakidae parasites, which can help improve the management and conservation of this important fish species. Abstract The Argentine hake (Merluccius hubbsi) is a vital fishery species in the Southwest Atlantic, recognized for its substantial economic importance. Previous studies have identified Anisakidae larvae as common parasites of M. hubbsi. However, the nutritional relationships between these parasites and their host remain poorly understood. This study employs stable isotope techniques to investigate the specific nutritional relationships between Anisakidae larvae and different tissues of M. hubbsi. The findings reveal notable differences in δ13C and δ15N compositions between the parasites and their host. The lower δ13C values in parasites compared to host tissues indicate the utilization of different carbon sources. The δ15N values of the parasites partially overlap with those of the host’s stomach, indicating that the parasites primarily derive nutrients from the host’s stomach. Nutritional niche indicators show that parasites have a broad carbon range (CR) and nitrogen range (NR), suggesting a high diversity in nutritional sources. The trophic discrimination factor (ΔTDF), which represents the difference in stable isotope values between host tissues and parasites, was analyzed for both δ13C and δ15N. The ΔTDFδ13C between the host liver and the parasites showed the greatest variation, indicating a strong dependence of the parasites on the liver’s carbon sources. In contrast, variations in ΔTDFδ15N between host tissues and parasites were minimal. Analyzing ΔTDF across different stages of gonadal maturity in the host fish indicates that, as the gonads of the host fish mature, ΔTDFδ13C between host tissues and parasites significantly decreases (p < 0.01). The Kruskal–Wallis test showed significant differences in ΔTDFδ13C values among different parasite infection levels in muscle, liver, and stomach tissues, while no significant differences were found for ΔTDFδ15N values. These findings offer valuable insights into the nutritional relationships between parasites and hosts, aiding in a better understanding of the growth conditions and habitats of M. hubbsi.


Introduction
The Argentine hake (Merluccius hubbsi), a member of the order Gadiformes and the family Merlucciidae, primarily inhabits the continental shelf of Uruguay and Argentina between 35 • S and 54 • S, typically at depths of 50 to 400 m [1].It is a crucial fishery species in this region, with an annual catch of approximately 415,000 tons, according to the FAO (2021) [2].M. hubbsi plays a vital role in the marine food web, feeding on small fish, crustaceans, and other marine invertebrates, while also serving as prey for larger predators such as seals, sharks, and large seabirds [3].Consequently, M. hubbsi plays a crucial role in maintaining the balance and functionality of marine ecosystems.The health of its population directly affects the ecological balance and the sustainable use of fishery resources.However, overfishing and environmental changes present significant challenges to the sustainability of this species.Consequently, there is an urgent need for in-depth studies on its ecological and biological characteristics to develop effective conservation and management measures [4,5].
Anisakidae are prevalent in marine environments worldwide and are among the most common parasites found in marine fish [6].The infection rates of these parasites are notably high.For instance, studies have reported infection rates of 83-85% in two species of mackerel along the Portuguese Atlantic coast [7].Similarly, research from the eastern coastal waters of Fujian Province, China, observed infection rates of 41.2%, with some species exhibiting rates as high as 100% [8].The significant impact of Anisakidae infections on fish growth and health is well-documented.Cod infected with Anisakidae larvae exhibit significantly reduced growth in both weight and length compared to uninfected individuals [9].Additionally, infected cod have shown compromised immune responses, leading to higher infection rates and increased mortality [10].Parasitic infections can also affect adversely reproductive capabilities, as evidenced by findings that such infections hinder gonadal development and decrease spawning amount, thereby impacting overall population reproductive rates [11].These results suggest that Anisakidae infections not only significantly affect fish health and growth, but also have important implications for population dynamics and ecosystem functionality.Despite extensive research on the prevalence and effects of Anisakidae on fish, studies on the specific nutritional relationships between these parasites and their hosts are limited.Understanding these nutritional dynamics is crucial for assessing the resource status of fish populations and developing sustainable fisheries management strategies.
Stable isotope analysis is a powerful ecological tool that can accurately trace the flow of carbon and nitrogen between different trophic levels, offering a unique perspective on energy transfer and material cycling between hosts and parasites [12,13].Research has shown that parasites often exhibit lower δ 15 N values, indicating a distinct nutritional strategy compared to conventional predator-prey relationships [14].Additionally, studies have demonstrated that different types of parasites and their hosts display unique isotopic signatures, reflecting the complexity of host-parasite nutrient flow [15].These findings underscore the significant potential of stable isotope analysis in uncovering interactions between parasites and their hosts.Despite the extensive use of stable isotope techniques to explore host-parasite interactions, research specifically addressing the nutritional relationships between M. hubbsi and its parasitic Anisakidae is still lacking.Understanding these relationships is crucial for assessing the ecological impact of parasitism on M. hubbsi and for developing informed fisheries management strategies.
This study aims to elucidate the nutritional relationships between Anisakidae and their host M. hubbsi, as well as the ecological significance of these interactions.By analyzing the effects of Anisakidae infections on the stable isotope composition and fractionation (differences in δ 13 C and δ 15 N between host tissues and parasites) in various tissues of M. hubbsi, we seek to provide a detailed understanding of these dynamics.Samples of M. hubbsi were collected from various locations and time periods.Stable isotope values of carbon and nitrogen were measured in the muscle, liver, and stomach tissues, as well as in the Anisakidae found within these hosts.Nutritional niche indicators for each tissue were calculated, and statistical analyses were conducted to examine the relationships between parasite infection levels, host gonadal maturity, and stable isotope fractionation.This research aims to offer scientific insights into the growth conditions and sustainable resource utilization of M. hubbsi.

Sample Collection Time and Locations
From October 2022 to February 2023, the samples of M. hubbsi were collected during trawling operations conducted by China's large trawlers in the high seas outside the exclusive economic zones of Argentina and the Falkland Islands (Malvinas).A total of 435 fish samples were collected.The samples were frozen at −20 • C and transported back to the laboratory.Once in the laboratory, the samples were thawed, and fork length and body weight were measured.Gonadal maturity was also assessed.Detailed information on the sampling dates, locations, and sample characteristics is provided in Table 1.

Gonadal Maturity Identification
Gonadal maturity stages were identified based on morphological characteristics as follows [16]: 1.
Stage I (Immature): Small, thin, and thread-like gonads with no visible differentiation; 2.
Stage II (Developing): Enlargement of gonads with visible oocytes in females and whitish testes in males; 3.
Stage III (Mature): Fully developed gonads with larger ovaries containing visible eggs in females and enlarged, whitish testes in males; 4.
Stage IV (Spawning): Maximum-size gonads with ovaries filled with mature eggs in females and large, milky testes in males.
Out of the 435 fish samples collected, 46 individuals were classified as having Stage I gonadal maturity, where it was not possible to distinguish between males and females due to the immature state of the gonads.The remaining fish had a sex ratio of approximately 2.5 females to 1 male (278 females and 111 males).

Collection and Identification of Parasites
A total of 435 samples of M. hubbsi were dissected to collect parasites from different infection sites.The parasites were marked and counted, then cleaned with distilled water and fixed in 70% ethanol solution, which was heated to around 70 • C. The fixed parasites were then transferred to a glycerin clearing solution to become transparent for subsequent microscopic observation [17].The cleared parasites were placed on microscope slides for observation and photographed for morphological identification.The body length and width of the parasites were measured.Morphological analysis was conducted according to the guidelines in Aquatic Animal Parasitology [18].Anisakidae third-stage larvae (L 3 ) were identified by their milky-white, coiled appearance with a blunt, rounded head and a boring tooth on the ventral side of the head.The tail is pointed, with most species possessing a terminal caudal process, except for members of the genus Contracaecum.The body length of Anisakidae L 3 ranges from 1.5 to 50.0 mm, and the body width ranges from 0.05 to 1.2 mm.Due to the limitations of morphological identification and the need for additional methods such as molecular biology for accurate species identification at the larval stage, the larvae in this study were identified only to the family level for further analysis.

Stable Isotope Analysis
From the 435 collected M. hubbsi samples, 75 were randomly selected for carbon and nitrogen stable isotope analysis.Muscle, liver, and stomach tissues, along with parasites from these 75 fish, were placed into 5 mL cryogenic vials and then freeze-dried for 24 h.Once dried, the samples were ground into a homogeneous powder using a ball mill.Approximately 1.50 ± 0.05 mg of each sample was weighed and wrapped in tin foil.The wrapped samples were then analyzed for carbon and nitrogen stable isotope ratios (δ 13 C and δ 15 N) using a Sercon Integra2 elemental analyzer-stable isotope ratio mass spectrometer (EA-IRMS).The relevant calculation formulas are as follows [19]: where X represents δ 13 C or δ 15 N and R sample is the measured isotope ratio ( 13 C/ 12 C or 15 N/ 14 N).The standard reference materials were Pee Dee Belemnite (PDB) for carbon and atmospheric nitrogen (N2-atm) for nitrogen.To ensure the accuracy of the experimental results, 3 blank samples were run before the actual samples, and, for every 12 samples, 2 standard samples were included.The standard materials used were IAEA-600 (δ 13 C = −27.771‰;δ 15 N = 1‰) and acetanilide (δ 13 C = −29.53‰;δ 15 N = 1.18‰).The analytical precision for δ 13 C and δ 15 N was ±0.2‰, and the stable isotope data are reported in parts per thousand (‰).

Trophic Niche Calculation
To evaluate the trophic niches of different tissues of M. hubbsi as well as the overall parasites infecting them, we analyzed several key indicators: carbon range (CR, representing the variation in δ 13 C values), nitrogen range (NR, representing the variation in δ 15 N values), total area (TA), standard ellipse area (SEA), and sample-size-corrected ellipse area (SEAc).

1.
CR: This is the range of δ 13 C values within a sample, representing the difference between the maximum and minimum δ 13 C values.The Trophic Discrimination Factor (∆TDF) represents the difference in δ 13 C or δ 15 N values between two trophic levels.In this study, ∆TDF is used to evaluate the stable isotope fractionation between different tissues of the host (M.hubbsi) and the parasites infecting them.This measure reflects the changes in isotopes as food transfers from one trophic level to the next, which is crucial for understanding the nutritional relationships and energy flow between the host and parasites.The relevant calculation formula is as follows [12]: where δX host tissue represents the stable isotope values (δ 13 C or δ 15 N) of specific host tissues and δX parasite represents the stable isotope values (δ 13 C or δ 15 N) of the parasites.

Data Processing
Scatter plots were generated using the ggplot2 package to visualize the distribution of δ 13 C and δ 15 N values across different host tissues (muscle, liver, stomach) and the parasites.Standard ellipses were added to represent the variation range for each sample type.The SIBER package was employed to calculate various trophic niche indicators, including carbon range (CR), nitrogen range (NR), total area (TA), standard ellipse area (SEA), and sample-size-corrected ellipse area (SEAc).Boxplots were created using the ggplot2 package, with significance annotations (p-values) derived from one-way ANOVA to compare the ∆TDF values (δ 13 C and δ 15 N) between different host tissues (muscle, liver, stomach) and parasites.Pairwise comparisons were conducted using Tukey's post hoc test to identify significant differences between specific tissue types.The parasite counts across different stages of host gonadal maturity were also compared using Kruskal-Wallis tests followed by Dunn's test with Bonferroni correction to identify significant differences between specific stages.The ∆TDF values (δ 13 C and δ 15 N) across the different stages of host gonadal maturity were compared using analysis of variance (ANOVA) followed by Tukey's HSD post hoc test to evaluate the effect of sexual maturity.Kruskal-Wallis tests and post hoc Mann-Whitney U tests were used to analyze the relationship between ∆TDF values (δ 13 C and δ 15 N) and parasite infection levels.All statistical analyses and visualizations were performed using R version 4.3.3.

Biological Characteristics of M. hubbsi
The fork length of the collected M. hubbsi samples ranged from 217 mm to 560 mm, with an average length of 316.26 ± 57.78 mm (Figure 1a).The body weight varied from 85 g to 1370 g, with an average weight of 278.97 ± 187.82 g (Figure 1b).Gonadal maturity was classified into four stages, with the majority of individuals in stage two (Figure 1c).To verify the statistical significance of the developmental stages of the fish, chi-squared tests were conducted.The results revealed significant differences in the distributions of fork length (X 2 = 189.36,df = 9, p < 0.0001) and body weight (X 2 = 205.43,df = 15, p < 0.0001) across developmental stages.
verify the statistical significance of the developmental stages of the fish, chi-squared tests were conducted.The results revealed significant differences in the distributions of fork length (Χ 2 = 189.36,df = 9, p < 0.0001) and body weight (Χ 2 = 205.43,df = 15, p < 0.0001) across developmental stages.

Parasite Infection in M. hubbsi
Microscopic examination of the collected parasites revealed a total of 8438 parasites from the 435 M. hubbsi samples, with an infection rate of 99.08% and an average infection intensity of 19.56 ± 24.76 parasites per fish.The infection intensity ranged from 0 parasites per fish (with four fish showing no infection) to a maximum of 256 parasites per fish.Macroscopically, the parasites appeared as milky-white, coiled forms.Under the microscope, the parasites exhibited a blunt rounded anterior end with a boring tooth at the apex, distinct transverse striations, and a caudal process (Figure 2), consistent with the morphology of Anisakidae larvae.The collected Anisakidae larvae ranged in length from 8.04 to 28.57 mm and in width from 0.14 to 0.66 mm, confirming them as third-stage larvae (L3) of the Anisakidae family.

Parasite Infection in M. hubbsi
Microscopic examination of the collected parasites revealed a total of 8438 parasites from the 435 M. hubbsi samples, with an infection rate of 99.08% and an average infection intensity of 19.56 ± 24.76 parasites per fish.The infection intensity ranged from 0 parasites per fish (with four fish showing no infection) to a maximum of 256 parasites per fish.Macroscopically, the parasites appeared as milky-white, coiled forms.Under the microscope, the parasites exhibited a blunt rounded anterior end with a boring tooth at the apex, distinct transverse striations, and a caudal process (Figure 2), consistent with the morphology of Anisakidae larvae.The collected Anisakidae larvae ranged in length from 8.04 to 28.57 mm and in width from 0.14 to 0.66 mm, confirming them as third-stage larvae (L3) of the Anisakidae family.
verify the statistical significance of the developmental stages of the fish, chi-squared tests were conducted.The results revealed significant differences in the distributions of fork length (Χ 2 = 189.36,df = 9, p < 0.0001) and body weight (Χ 2 = 205.43,df = 15, p < 0.0001) across developmental stages.

Parasite Infection in M. hubbsi
Microscopic examination of the collected parasites revealed a total of 8438 parasites from the 435 M. hubbsi samples, with an infection rate of 99.08% and an average infection intensity of 19.56 ± 24.76 parasites per fish.The infection intensity ranged from 0 parasites per fish (with four fish showing no infection) to a maximum of 256 parasites per fish.Macroscopically, the parasites appeared as milky-white, coiled forms.Under the microscope, the parasites exhibited a blunt rounded anterior end with a boring tooth at the apex, distinct transverse striations, and a caudal process (Figure 2), consistent with the morphology of Anisakidae larvae.The collected Anisakidae larvae ranged in length from 8.04 to 28.57 mm and in width from 0.14 to 0.66 mm, confirming them as third-stage larvae (L3) of the Anisakidae family.Through dissection, Anisakidae larvae were found in various parts of M. hubbsi, including muscle, liver, stomach, intestine, pyloric caeca, and gonads.Detailed statistics showed that the primary infection sites were the muscle, liver, and stomach, with 3669 parasites (43.51%), 3316 parasites (39.30%), and 1446 parasites (17.14%) detected in these tissues, respectively.Other parts (intestine, pyloric caeca, and gonads) contained only 7 parasites in total, accounting for 0.05% of the total.Overall, 91.72% of the sampled fish had Anisakidae larvae in their muscle, 88.51% in their liver, and 76.55% in their stomach.Therefore, this study focuses primarily on the major infection sites: muscle, liver, and stomach.
The analysis of the number of parasites infecting M. hubbsi revealed the distribution of parasite infection (Figure 3).Most of the fish samples were infected with 1 to 20 parasites, with the highest proportion of fish (44.37%) being infected with 1 to 10 parasites.
Biology 2024, 13, x FOR PEER REVIEW 7 of 15 Through dissection, Anisakidae larvae were found in various parts of M. hubbsi, including muscle, liver, stomach, intestine, pyloric caeca, and gonads.Detailed statistics showed that the primary infection sites were the muscle, liver, and stomach, with 3669 parasites (43.51%), 3316 parasites (39.30%), and 1446 parasites (17.14%) detected in these tissues, respectively.Other parts (intestine, pyloric caeca, and gonads) contained only 7 parasites in total, accounting for 0.05% of the total.Overall, 91.72% of the sampled fish had Anisakidae larvae in their muscle, 88.51% in their liver, and 76.55% in their stomach.Therefore, this study focuses primarily on the major infection sites: muscle, liver, and stomach.
The analysis of the number of parasites infecting M. hubbsi revealed the distribution of parasite infection (Figure 3).Most of the fish samples were infected with 1 to 20 parasites, with the highest proportion of fish (44.37%) being infected with 1 to 10 parasites.The results of the average parasite count in M. hubbsi with different gonad maturity stages (Table 2) indicate that, as gonad maturity increases, the average number of parasites in the fish samples also significantly increases.Individuals with higher gonad maturity stages tend to have more parasites.A Kruskal-Wallis test was conducted to compare the parasite counts among the four different gonad maturity stages.The test revealed a statistically significant difference in parasite counts between the different stages (H = 102.88,p < 0.001).Post hoc comparisons using Dunn's test with Bonferroni correction indicated the following significant differences: the parasite count in stage IV was significantly higher than in stages I, II, and III (p < 0.001); additionally, the parasite count in stage III was significantly higher than in stages I (p < 0.001) and II (p < 0.001), and the parasite count in stage II was marginally higher than in stage I (p = 0.0598, adjusted p = 0.359).

Stable Isotope Composition
The stable isotope analysis indicated distinct δ 13 C and δ 15 N values for the parasites and various host tissues.The δ 13 C values of the parasites spanned from −22.10‰ to −18.51‰, with a range difference of 3.59‰, while the δ 15 N values ranged from 10.45‰ to 14.54‰, with a range difference of 4.09‰.For the host tissues, the δ 13 C values were observed as follows: muscle ranged from −19.43‰ to −17.86‰ (range difference: 1.57‰),The results of the average parasite count in M. hubbsi with different gonad maturity stages (Table 2) indicate that, as gonad maturity increases, the average number of parasites in the fish samples also significantly increases.Individuals with higher gonad maturity stages tend to have more parasites.A Kruskal-Wallis test was conducted to compare the parasite counts among the four different gonad maturity stages.The test revealed a statistically significant difference in parasite counts between the different stages (H = 102.88,p < 0.001).Post hoc comparisons using Dunn's test with Bonferroni correction indicated the following significant differences: the parasite count in stage IV was significantly higher than in stages I, II, and III (p < 0.001); additionally, the parasite count in stage III was significantly higher than in stages I (p < 0.001) and II (p < 0.001), and the parasite count in stage II was marginally higher than in stage I (p = 0.0598, adjusted p = 0.359).

Stable Isotope Composition
The stable isotope analysis indicated distinct δ 13 C and δ 15 N values for the parasites and various host tissues.The δ 13 C values of the parasites spanned from −22.10‰ to −18.51‰, with a range difference of 3.59‰, while the δ 15 N values ranged from 10.45‰ to 14.54‰, with a range difference of 4.09‰.For the host tissues, the δ 13 C values were observed as follows: muscle ranged from −19.43‰ to −17.86‰ (range difference: 1.57‰), liver from −21.49‰ to −17.32‰ (range difference: 4.17‰), and stomach from −20.72‰ to −16.41‰ (range difference: 4.31‰).The δ 15 N values for muscle, liver, and stomach varied from 11.59‰ to 16.49‰ (range difference: 4.90‰), 13.40‰ to 17.28‰ (range difference: 3.88‰), and 11.92‰ to 16.24‰ (range difference: 4.32‰), respectively.Figure 4 shows that the confidence ellipse for the stable isotope composition of the parasites minimally overlaps with those of the host muscle and liver, but partially overlaps with the stomach.4 shows that the confidence ellipse for the stable isotope composition of the parasites minimally overlaps with those of the host muscle and liver, but partially overlaps with the stomach.The analysis revealed that the parasites had a carbon range (CR) of 3.88 and a nitrogen range (NR) of 4.17.For the host tissues of M. hubbsi, the CR and NR values were as follows: muscle (CR: 4.90, NR: 1.57), liver (CR: 4.32, NR: 3.95), and stomach (CR: 4.09, NR: 3.59).The total area (TA) values revealed that the stomach (10.22) and parasites (9.38) occupied broader niche widths, whereas the host muscle exhibited the narrowest niche width (TA: 5.44).Furthermore, the standard ellipse area (SEA) and its confidence interval (SEAc) for the parasites were 1.41 and 1.43, respectively.These values were comparable to those for the host liver (SEA: 1.54, SEAc: 1.56) and stomach (SEA: 1.78, SEAc: 1.80), but were higher than those for the host muscle, which had the lowest SEA and SEAc values (0.92 and 0.93) (Table 3).

ΔTDF and Correlation Analysis
The analysis of the trophic discrimination factor (ΔTDF), representing the difference in stable isotope values (δ 13 C and δ 15 N) between host tissues (M.hubbsi muscle, liver, stomach) and the infecting Anisakidae larvae within the same host individuals, revealed distinct patterns.The   13  values showed considerable variation.Specifically, the liver and stomach exhibited the greatest ranges in   13  , indicating a wide range of carbon isotope discrimination between these tissues and the parasites.In contrast, the muscle tissue exhibited the smallest range in   13  .For nitrogen isotope fractionation The analysis revealed that the parasites had a carbon range (CR) of 3.88 and a nitrogen range (NR) of 4.17.For the host tissues of M. hubbsi, the CR and NR values were as follows: muscle (CR: 4.90, NR: 1.57), liver (CR: 4.32, NR: 3.95), and stomach (CR: 4.09, NR: 3.59).The total area (TA) values revealed that the stomach (10.22) and parasites (9.38) occupied broader niche widths, whereas the host muscle exhibited the narrowest niche width (TA: 5.44).Furthermore, the standard ellipse area (SEA) and its confidence interval (SEAc) for the parasites were 1.41 and 1.43, respectively.These values were comparable to those for the host liver (SEA: 1.54, SEAc: 1.56) and stomach (SEA: 1.78, SEAc: 1.80), but were higher than those for the host muscle, which had the lowest SEA and SEAc values (0.92 and 0.93) (Table 3).

∆TDF and Correlation Analysis
The analysis of the trophic discrimination factor (∆TDF), representing the difference in stable isotope values (δ 13 C and δ 15 N) between host tissues (M.hubbsi muscle, liver, stomach) and the infecting Anisakidae larvae within the same host individuals, revealed distinct patterns.The ∆TDF δ 13 C values showed considerable variation.Specifically, the liver and stomach exhibited the greatest ranges in ∆TDF δ 13 C , indicating a wide range of carbon isotope discrimination between these tissues and the parasites.In contrast, the muscle tissue exhibited the smallest range in ∆TDF δ 13 C .For nitrogen isotope fractionation (∆TDF δ 15 N ), the ranges of ∆TDF across the three host tissues were minimal.Additionally, significant differences in ∆TDF δ 13 C were observed among the different tissues (p < 0.01).To identify these differences, we employed one-way ANOVA followed by Tukey's post-hoc test.The results indicated significant differences in ∆TDF δ 15 N between muscle and liver, and between liver and stomach (p < 0.01), whereas the difference between stomach and muscle was not significant (Figure 5).
Biology 2024, 13, x FOR PEER REVIEW 9 of 15 (  15  ), the ranges of ΔTDF across the three host tissues were minimal.Additionally, significant differences in   13  were observed among the different tissues (p < 0.01).
To identify these differences, we employed one-way ANOVA followed by Tukey's posthoc test.The results indicated significant differences in   15  between muscle and liver, and between liver and stomach (p < 0.01), whereas the difference between stomach and muscle was not significant (Figure 5).The comparisons of ΔTDF values across the different stages of gonadal maturity revealed that gonadal maturity significantly affects ΔTDF in various tissues (Table 4).As gonadal maturity increased,   13  values between host tissues (muscle, liver, stomach) and parasites significantly decreased (p < 0.01).Individuals in stage I of gonadal maturity exhibited the highest   13  values, whereas those in stages III and IV showed the lowest values.In contrast,   15  values for muscle, liver, and stomach did not show significant differences among different gonadal maturity groups (p > 0.05).The Kruskal-Wallis test results indicated significant differences in   13  values among different parasite load groups in muscle (p < 0.001), liver (p < 0.001), and stomach (p < 0.001) tissues.Post hoc pairwise comparisons using the Mann-Whitney U test revealed significant differences between muscle vs. liver (p < 0.001), muscle vs. stomach (p < 0.001), and liver vs. stomach (p < 0.001).For   15  , no significant differences were observed among different parasite load groups in any tissue (p > 0.05).The comparisons of ∆TDF values across the different stages of gonadal maturity revealed that gonadal maturity significantly affects ∆TDF in various tissues (Table 4).As gonadal maturity increased, ∆TDF δ 13 C values between host tissues (muscle, liver, stomach) and parasites significantly decreased (p < 0.01).Individuals in stage I of gonadal maturity exhibited the highest ∆TDF δ 13 C values, whereas those in stages III and IV showed the lowest values.In contrast, ∆TDF δ 15 N values for muscle, liver, and stomach did not show significant differences among different gonadal maturity groups (p > 0.05).The Kruskal-Wallis test results indicated significant differences in ∆TDF δ 13 C values among different parasite load groups in muscle (p < 0.001), liver (p < 0.001), and stomach (p < 0.001) tissues.Post hoc pairwise comparisons using the Mann-Whitney U test revealed significant differences between muscle vs. liver (p < 0.001), muscle vs. stomach (p < 0.001), and liver vs. stomach (p < 0.001).For ∆TDF δ 15 N , no significant differences were observed among different parasite load groups in any tissue (p > 0.05).

Infection Status of M. hubbsi by Anisakidae
In this study, the infection rate of M. hubbsi by Anisakidae reached 99.08%.The sampling area covered the high seas outside the exclusive economic zones of Argentina and the Falkland Islands (Malvinas), located on the Patagonian Shelf in the South Atlantic Ocean.This shelf is one of the largest and most productive ecosystems in the Southern Hemisphere [23].Influenced by the Falkland Current, the temperature in the Falkland Islands (Malvinas) decreases with increasing latitude, and Anisakidae are well-adapted to these cooler waters [24].Consequently, within the study area, the infection rate of Anisakidae larvae in M. hubbsi increases with latitude, reaching up to 100% between 44 • S and 46 • S [25].This high infection rate reflects the adaptability of Anisakidae to their environment.Furthermore, Anisakidae not only adapt well to low-temperature environments but also have a complex lifecycle that includes multiple host stages, which increases their survival opportunities in diverse ecosystems.Studies have shown that Anisakidae larvae primarily parasitize marine fish, while adult worms typically inhabit seabirds and marine mammals [26].This complex lifecycle allows Anisakidae to thrive and reproduce in various environments, enhancing their transmission potential.Additionally, the high infection rate of Anisakidae is also related to the ecological behavior of M. hubbsi.The different developmental stages of fish exhibit significant variations in physiological status, including growth patterns, immune function, and energy allocation, which influence their susceptibility to parasitic infections [27].The results from the Kruskal-Wallis test and Dunn's post hoc comparisons confirmed that there are significant differences in parasite counts among the different gonad maturity stages.Specifically, the parasite count in stage IV was significantly higher than in stages I, II, and III.Additionally, the parasite count in stage III was significantly higher than in stages I and II.These findings suggest that, as the gonad maturity increases, the parasite load also increases.This is likely due to the significant energy expenditure during reproduction, which temporarily weakens their immune system.Studies by Chaves-Pozo et al. have demonstrated that reproductive activities significantly impact fish immune function, making them more susceptible to parasitic infections [28].
Analysis of Anisakidae infection in M. hubbsi indicates that muscle, liver, and stomach are the primary infection sites, showing the parasites' preference for specific tissues, which is closely related to their life cycle.Anisakidae typically have a complex life cycle that includes multiple host stages.In the larval stage, Anisakidae primarily parasitize the stomach and intestines of fish because these organs directly contact with ingested food, providing ample nutrition for larvae [26].As the parasites develop, they migrate to other host tissues, which are rich in nutrients, supporting further growth and development [29].Muscle tissue, being the primary locomotive tissue of fish, is rich in proteins and energy, serving as an important nutritional source for parasites.The liver, being the center of metabolic activity, stores large amounts of glycogen and fats, which the parasites can utilize [30].These tissue characteristics make them ideal sites for parasitization.Furthermore, the high infection rate within the host may also be related to the parasite's mechanisms for evading the host's immune system.Studies have shown that Anisakidae can evade host immune defenses by secreting specific immunosuppressive substances, allowing them to survive and complete their lifecycle within the host [31].These adaptation and evasion mechanisms allow parasites to survive and proliferate within specific host tissues.

Stable Isotope Composition and Nutritional Niche Indicators
Stable isotope analysis revealed significant differences in δ 13 C and δ 15 N compositions between the tissues of M. hubbsi and their parasitic Anisakidae, reflecting different nutritional uptake and metabolic strategies.Hosts typically obtain carbon sources from primary producers or the other levels of the food chain, whereas parasites rely on carbon sources within the host, which are often more readily metabolized and circulated organic substances [32].Consequently, the δ 13 C values of parasites are relatively lower than those of the host tissues.Moreover, the δ 15 N range of parasites is narrower and partially overlaps with the δ 15 N values of the host's stomach tissues.This observation is consistent with Poulin and Thomas's research, which found that the δ 15 N values of parasites often resemble those of the host's digestive system tissues [33].The digestive system of fish generally consists of four main parts: the mouth, pharynx, stomach, and intestines.After ingestion, food undergoes initial processing in the mouth and pharynx, followed by further mechanical and chemical digestion in the stomach.Digestive fluids and enzymes in the stomach break down food into absorbable nutrients, which are then absorbed into the host's bloodstream through the intestines.Parasites attach to various tissues and obtain nutrients through their surface or specialized absorption structures from the host's digestive system or other tissues.This leads to the similarity in δ 15 N values between parasites and the host's digestive system tissues, as parasites directly obtain their nitrogen sources from these tissues [34].
Nutritional niche indicators further validated the stable isotope composition results, revealing differences in trophic levels and niche breadth between parasites and host tissues.Higher NR values are typically associated with higher trophic levels and a broader range of food sources [22].The host muscle exhibited lower CR and NR values, indicating a more uniform nutritional source, whereas the liver and stomach showed higher CR and NR values, reflecting the complexity of their nutritional metabolism.The larger CR and NR values of Anisakidae might be due to their ability to obtain nutrients not only from the stomach contents of the host but also from other tissues such as the liver.The liver, being a vital organ for metabolism and storage, is rich in fats, glycogen, and proteins.By absorbing these stored substances, the parasites can access a diverse range of nutritional sources, which include different isotopic compositions, resulting in higher carbon and nitrogen range values [35].Furthermore, the metabolic mechanisms of the parasites may also contribute to their larger CR and NR values.Parasites possess efficient metabolic pathways that allow them to rapidly utilize various nutrients provided by the host.This efficient metabolism enables the parasites to exhibit significant differences in trophic levels and adapt to the nutritional resources available in different host tissues [36].Additionally, the high total area (TA) values of the stomach and parasites indicate a broader niche width, reflecting their diversity in food sources.The standardized ellipse area (SEA) and its confidence interval (SEAc) for parasites are similar to those of the host's liver and stomach, whereas the host muscle exhibits the lowest SEA and SEAc values.These results align with the stable isotope composition analysis, highlighting the parasites' reliance on nutrients from the host's stomach while also utilizing the host's own nutrients.This further demonstrates the close nutritional and metabolic relationship between the parasites and their host.

Stable Isotope Fractionation and Correlation Analysis
The carbon isotope fractionation results for different tissues of M. hubbsi indicated that the liver exhibited the largest variation in ∆TDF δ 13 C , while the muscle tissue showed the smallest variation.This suggests significant differences in how parasites utilize the nutrients in the host's liver tissue.Lafferty and Kuris's research also supports the finding that parasites often exhibit significant isotope fractionation in highly metabolic tissues like the liver, consistent with our study [37].The liver is a highly metabolic organ, rich in fats, glycogen, and proteins [38].Parasites, while absorbing and utilizing these stored substances, exhibit more pronounced carbon isotope fractionation through complex metabolic pathways such as glycolysis, gluconeogenesis, and fatty acid metabolism [39].On the other hand, the nitrogen isotope fractionation results for different tissues of M. hubbsi showed minimal variation across the three host tissues.This consistency indicates that the nitrogen isotope fractionation by parasites is relatively uniform across different tissues.This may be because parasites rely mainly on amino acid metabolism and protein synthesis for their nitrogen metabolism, processes that are similar across different tissues [40].These findings are in line with the results of Marcogliese and Cone, who also reported consistent nitrogen isotope fractionation by parasites across different host tissues [41].
The comparison of ∆TDF across different gonadal maturity stages revealed that, with increasing gonadal maturity, the ∆TDF δ 13 C values for muscle, liver, and stomach tissues significantly decreased.This indicates that the difference in δ 13 C values between host tissues and parasites becomes smaller as the gonads mature.During gonadal development, the host reallocates nutrients to prioritize gonadal development, leading to a reduced nutrient supply for other tissues.Consequently, this affects the isotope fractionation effects between the host tissues and parasites [42].As gonadal development progresses, the host's metabolic demands increase, which may accelerate the metabolic utilization of carbon isotopes, thereby reducing δ 13 C values in host tissues and bringing them closer to those of the parasites [43].The roles and significance of different tissues in metabolic processes also affect isotope fractionation.For example, the liver, as a major organ for metabolism and detoxification, shows a significant decrease in ∆TDF δ 13 C , reflecting increased efficiency in carbon isotope utilization under high metabolic states [44].In the early stages of gonadal development (stage I), the host's metabolic activity is focused on growth and weight gain, providing ample nutrient supply to muscle, liver, and stomach tissues, resulting in higher ∆TDF δ 13 C values.As gonadal development advances (stages III and IV), the host reallocates nutrients to support gonadal development, reducing the nutrient supply to other tissues and consequently lowering ∆TDF δ 13 C values [45].In contrast to δ 13 C isotope fractionation, there were no significant differences in ∆TDF δ 15 N values for muscle, liver, and stomach tissues across different gonadal maturity groups.This may be related to the crucial role of these tissues in protein synthesis, as proteins are essential components of organisms, and their synthesis and metabolic processes remain relatively constant across different gonadal maturity stages [44].
The Kruskal-Wallis test results revealed significant differences in ∆TDF δ 13 C values among different levels of parasite infection in muscle, liver, and stomach tissues, indicating that parasite load significantly affects the trophic positions in these tissues.Post hoc pairwise comparisons using the Mann-Whitney U test confirmed significant differences between muscle vs. liver, muscle vs. stomach, and liver vs. stomach.Host muscle tissue, primarily composed of proteins, may contain a higher proportion of lighter carbon isotopes.As the number of parasites increases, they extensively consume these proteins, leading to preferential utilization of the lighter carbon isotopes in the host muscle, thereby reducing the δ 13 C values and lowering ∆TDF δ 13 C values [46].The efficient nutrient absorption by parasites in the stomach tissue is likely due to the stomach's abundance of easily digestible and absorbable nutrients, such as proteins and fats, enabling parasites to quickly utilize these nutrients for metabolism and growth [47].In the liver, although the differences were significant, the relationship remains complex.The liver is a crucial organ for metabolism and storage, participating in various metabolic pathways such as gluconeogenesis, fat metabolism, and protein synthesis, all of which can influence isotope fractionation effects [48].In contrast, the Kruskal-Wallis analysis for nitrogen isotope fractionation showed no significant differences in ∆TDF δ 15 N values across all host tissues at different levels of parasite infection.This might be because the nitrogen demand by parasites remains relatively stable at different infection levels, and their utilization of nitrogen sources does not significantly change with increasing infection numbers [49].

Conclusions
This study utilized stable isotope techniques to deeply analyze the impact of Anisakidae infection on M. hubbsi, revealing specific nutritional relationships between the parasite and the host.The results showed that the infection rate of Anisakidae in M. hubbsi reached 99.08%, with an average of 19.56 parasites per fish.The δ 13 C values of the parasites were significantly lower than those of the host, while the δ 15 N values of the parasites partially overlapped with those of the host's stomach tissues.Additionally, the carbon range (CR) and nitrogen range (NR) of the parasites were both large.The study also found that the ∆TDF δ 13 C showed the greatest variation in the host's liver.As the gonadal maturity of the host increased, the ∆TDF δ 13 C values in all host tissues significantly decreased.Our findings also indicate that higher levels of parasite infection significantly affect ∆TDF δ 13 C values in muscle, liver, and stomach tissues, while no significant differences were observed in ∆TDF δ 15 N values.
Future research should consider examining stable isotope composition variations of parasites and hosts under different environmental conditions to understand the broader ecological impacts.While this study primarily focused on the stable isotope analysis of carbon and nitrogen in different tissues of M. hubbsi and its parasitic Anisakidae, it is important to acknowledge that understanding the full impact of parasitic infection on host physiology and immune function across various developmental stages was beyond our scope.However, these aspects are crucial for a comprehensive understanding of host-parasite interactions.Long-term studies assessing interannual variations in parasitic infection rates and stable isotope compositions will further elucidate these dynamics.Integrating molecular biology techniques such as genomic and transcriptomic analyses in future studies could uncover detailed metabolic pathways and interaction mechanisms, clarifying the effects of parasites on host nutrition and health at the molecular level.These suggestions aim to build on the findings of this study, providing deeper insights and supporting sustainable fisheries management strategies for M. hubbsi.

Figure 4 .
Figure 4. δ 13 C and δ 15 N isotopic composition of different host tissues and Anisakidae in M. hubbsi.Dashed ellipses represent the isotopic distribution ranges for each sample type.

Figure 4 .
Figure 4. δ 13 C and δ 15 N isotopic composition of different host tissues and Anisakidae in M. hubbsi.Dashed ellipses represent the isotopic distribution ranges for each sample type.

Figure 5 .
Figure 5. Box plots of ΔTDF values between host tissues of M. hubbsi and their infecting Anisakidae larvae.(a)   13  values showing the difference in δ 13 C between host tissues (muscle, liver, stomach) and the parasites; (b)   15  values showing the difference in δ 15 N between host tissues (muscle, liver, stomach) and the parasites.Asterisks indicate significance levels: **** for (p < 0.0001), *** for (p < 0.001), ns for not significant.The dots in subfigure (b) represent outlier data points for the   15  values between the host tissues (muscle, liver, stomach) and the parasites.

Figure 5 .
Figure 5. Box plots of ∆TDF values between host tissues of M. hubbsi and their infecting Anisakidae larvae.(a) ∆TDF δ 13 C values showing the difference in δ 13 C between host tissues (muscle, liver, stomach) and the parasites; (b) ∆TDF δ 15 N values showing the difference in δ 15 N between host tissues (muscle, liver, stomach) and the parasites.Asterisks indicate significance levels: **** for (p < 0.0001), *** for (p < 0.001), ns for not significant.The dots in subfigure (b) represent outlier data points for the ∆TDF δ 15 N values between the host tissues (muscle, liver, stomach) and the parasites.

Table 1 .
Sample size, length range, and weight range of M. hubbsi for different sampling dates and locations.

Table 3 .
Isotopic niche metrics for different host tissues and Anisakidae in M. hubbsi.

Table 3 .
Isotopic niche metrics for different host tissues and Anisakidae in M. hubbsi.

Table 4 .
The effect of gonad maturity on ΔTDF values (Mean ± SD) in different tissues of M. hubbsi.

Table 4 .
The effect of gonad maturity on ∆TDF values (Mean ± SD) in different tissues of M. hubbsi.